ABSTRACT
Glutathione-S-transferase (GST)-fusion proteins have become an effective reagent to use in the study of protein-protein interactions. GST-fusion proteins can be produced in bacterial and mammalian cells in large quantities and purified rapidly. GST can be coupled to a glutathione matrix, which permits its use as an effective affinity column to study interactions in vitro or to purify protein complexes in cells expressing the GST-fusion protein. Here, we provide a technical description of the utilization of GST-fusion proteins as both a tool to study protein-protein interactions and also as a means to purify interacting proteins.
Subject(s)
Oncogene Proteins v-raf/chemistry , Protein Interaction Mapping/methods , Protein Interaction Maps , ras Proteins/chemistry , Antibodies/chemistry , Antibodies/immunology , Binding Sites , Chromatography, Affinity , Glutathione Transferase/chemistry , Glutathione Transferase/immunology , Protein BindingABSTRACT
We describe the design, synthesis, and optimization of a series of new inhibitors of V-RAF murine sarcoma viral oncogene homologue B1 (BRAF), a kinase whose mutant form (V600E) is implicated in several types of cancer, with a particularly high frequency in melanoma. Our previously described inhibitors with a tripartite A-B-C system (where A is a hinge binding pyrido[4,5-b]imidazolone system, B is an aryl spacer group, and C is a heteroaromatic group) were potent against purified (V600E)BRAF in vitro but were less potent in accompanying cellular assays. Substitution of different aromatic heterocycles for the phenyl based C-ring is evaluated herein as a potential means of improving the cellular potencies of these inhibitors. Substituted pyrazoles, particularly 3-tert-butyl-1-aryl-1H-pyrazoles, increase the cellular potencies without detrimental effects on the potency on isolated (V600E)BRAF. Thus, compounds have been synthesized that inhibit, with low nanomolar concentrations, (V600E)BRAF, its downstream signaling in cells [as measured by the reduction of the phosphorylation of extracellular regulated kinase (ERK)], and the proliferation of mutant BRAF-dependent cells. Concomitant benefits are good oral bioavailability and high plasma concentrations in vivo.
Subject(s)
Drug Design , Oncogene Proteins v-raf/chemistry , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Sarcoma Viruses, Murine/enzymology , Sequence Homology , Animals , Cell Line, Tumor , Female , Humans , Inhibitory Concentration 50 , Mice , Models, Molecular , Molecular Conformation , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/metabolism , Structure-Activity RelationshipABSTRACT
Durante el desarrollo del sistema nervioso de vertebrados, múltiples procesosfisiológicos participan en la generación de su compleja arquitectura celular yfuncionalidad. Entre ellos, la muerte celular programada que afecta a neuronas deconexión está reconocido como un proceso fundamental. Por otro lado, hay escasainformación disponible acerca de la muerte celular que afecta a célulasneuroepiteliales y a neuronas y glía recién nacidas, lo que impide que tengamosuna noción completa sobre el desarrollo neural. Los estudios de nuestro laboratoriohan demostrado que la muerte celular programada se encuentra finamente reguladay ocurre en etapas tan tempranas del desarrollo como la neurulación o laneurogénesis. Hemos caracterizado el papel que moléculas de supervivencia, comola proinsulina/insulina, c-Raf o HSC70, desempeñan bloqueando la apoptosisdependiente de caspasas, proceso que afecta a células neuroepitelialesproliferativas, así como a la generación de las células ganglionares de la retina. Esmás, la caracterización de estas señales fisiológicas originadas durante laneurogénesis de la retina nos ha proporcionado una nueva herramienta terapéuticapotencial para el tratamiento y atenuación de las neurodegeneraciones retinianas
During the development of the vertebrate nervous system, multiple physiologicalprocesses are involved in the generation of its complex cytoarchitecture andfunctionality. Among them, programmed cell death has been recognized as a keyprocess that affects connecting neurons. By contrast, there is limited informationavailable regarding the cell death that affects neuroepithelial cells, and recentlyborn neurons and glia, hindering the comprehensive understanding of neuraldevelopment. We have demonstrated that exquisitely regulated PCD occurs duringearly stages of neural development such as neurulation and neurogenesis. We havecharacterized how survival signals from proteins like proinsulin/insulin, c-Raf, andHSC70 counteract caspase-dependent apoptosis, which affects neuroepithelial cellsproliferation and the generation of retinal ganglion cells. Furthermore, the characterization of these physiological signals during retinal neurogenesis has thepotential to provide new therapeutic tools to attenuate retinal neurodegeneration
Subject(s)
Nervous System/chemistry , Nervous System , Cell Death , Cell Death/physiology , Insulin/chemical synthesis , Insulin/pharmacology , Proinsulin/chemistry , Caspases/chemistry , Caspases/chemical synthesis , Neurons/chemistry , Insulin/chemistry , Apoptosis , Eutrophication , Cell Differentiation , Retina/chemistry , Retina , Proto-Oncogene Proteins c-raf/chemistry , Oncogene Proteins v-raf/chemistry , Oncogene Proteins v-raf/chemical synthesisABSTRACT
To evaluate the timing of mutations in BRAF (v-raf murine sarcoma viral oncogene homolog B1) during melanocytic neoplasia, we carried out mutation analysis on microdissected melanoma and nevi samples. We observed mutations resulting in the V599E amino-acid substitution in 41 of 60 (68%) melanoma metastases, 4 of 5 (80%) primary melanomas and, unexpectedly, in 63 of 77 (82%) nevi. These data suggest that mutational activation of the RAS/RAF/MAPK pathway in nevi is a critical step in the initiation of melanocytic neoplasia but alone is insufficient for melanoma tumorigenesis.
